title: Hyperglycemia induces differential change in oxidative stress at gene expression and functional levels in HUVEC and HMVEC.
abstract: BACKGROUND
  Endothelial dysfunction precedes pathogenesis of vascular complications in diabetes. In recent years, the mechanisms of endothelial dysfunction were investigated to outline strategies for its treatment. However, the therapies for dysfunctional endothelium resulted in multiple clinical trial failures and remain elusive. There is a need for defining hyperglycemia-induced endothelial dysfunction with both generic and specific dysfunctional changes in endothelial cells (EC) using a systems approach. In this study, we investigated hyperglycemia-induced endothelial dysfunction in HUVEC and HMVEC. We investigated hyperglycemia-induced functional changes (superoxide (O₂⁻), and hydrogen peroxide (H₂O₂) production and mitochondrial membrane polarization) and gene expression fingerprints of related enzymes (nitric oxide synthase, NAD(P)H oxidase, and reactive oxygen species (ROS) neutralizing enzymes) in both ECs.
  
  
  METHODS
  Gene expression of NOS2, NOS3, NOX4, CYBA, UCP1, CAT, TXNRD1, TXNRD2, GPX1, NOX1, SOD1, SOD2, PRDX1, 18s, and RPLP0 were measured using real-time PCR. O₂⁻ production was measured with dihydroethidium (DHE) fluorescence measurement. H2O2 production was measured using Amplex Red assay. Mitochondrial membrane polarization was measured using JC-10 based fluorescence measurement.
  
  
  RESULTS
  We showed that the O₂⁻ levels increased similarly in both ECs with hyperglycemia. However, these endothelial cells showed significantly different underlying gene expression profile, H₂O₂ production and mitochondrial membrane polarization. In HUVEC, hyperglycemia increased H₂O₂ production, and hyperpolarized mitochondrial membrane. ROS neutralizing enzymes SOD2 and CAT gene expression were downregulated. In contrast, there was an upregulation of nitric oxide synthase and NAD(P)H oxidase and a depolarization of mitochondrial membrane in HMVEC. In addition, ROS neutralizing enzymes SOD1, GPX1, TXNRD1 and TXNRD2 gene expression were significantly upregulated in high glucose treated HMVEC.
  
  
  CONCLUSIONS
  Our findings highlighted a unique framework for hyperglycemia-induced endothelial dysfunction. We showed that multiple pathways are differentially affected in these endothelial cells in hyperglycemia. High occurrences of gene expression changes in HMVEC in this study supports the hypothesis that microvasculature precedes macrovasculature in epigenetic regulation forming vascular metabolic memory. Identifying genomic phenotype and corresponding functional changes in hyperglycemic endothelial dysfunction will provide a suitable systems biology approach for understanding underlying mechanisms and possible effective therapeutic intervention.
authors.affiliation: Department of Biomedical Engineering, Wayne State University, 2322 Engineering, 5050 Anthony Wayne Dr,, Detroit, MI 48202, USA. hemang@wayne.edu.
authors.forename: Hemang
authors.initials: H
authors.lastname: Patel
authors.forename: Juan
authors.initials: J
authors.lastname: Chen
authors.forename: Kumuda C
authors.initials: KC
authors.lastname: Das
authors.forename: Mahendra
authors.initials: M
authors.lastname: Kavdia
mesh_terms: D002478:Cells, Cultured; D042783:Endothelial Cells / Q000201:enzymology*; D015971:Gene Expression Regulation, Enzymologic; D005947:Glucose / Q000378:metabolism*; D061307:Human Umbilical Vein Endothelial Cells / Q000201:enzymology*; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism; D006943:Hyperglycemia / Q000201:enzymology* / Q000235:genetics; D007473:Ion Channels / Q000235:genetics; D053078:Membrane Potential, Mitochondrial; D055806:Microvessels / Q000201:enzymology*; D024101:Mitochondrial Proteins / Q000235:genetics; D019255:NADPH Oxidases / Q000235:genetics; D051267:NF-E2-Related Factor 2 / Q000235:genetics; D019001:Nitric Oxide Synthase / Q000235:genetics; D018384:Oxidative Stress / Q000235:genetics*; D012333:RNA, Messenger / Q000378:metabolism; D013481:Superoxides / Q000378:metabolism; D049490:Systems Biology; D013997:Time Factors; D000071256:Uncoupling Protein 1
chemical_list: D007473:Ion Channels; D024101:Mitochondrial Proteins; D051267:NF-E2-Related Factor 2; C495635:NFE2L2 protein, human; D012333:RNA, Messenger; C000604371:UCP1 protein, human; D000071256:Uncoupling Protein 1; D013481:Superoxides; D006861:Hydrogen Peroxide; D019001:Nitric Oxide Synthase; D019255:NADPH Oxidases; D005947:Glucose